A widely used technique for separation of biological macromolecules in solution (macrosolutes). known as isopycnic density gradient separation, involves high-speed centrifugation of the macrosolutes at relatively low concentration together with a small solute, referred to as the gradient-forming solute at high concentration. The density gradient, arising from the concentration gradient of the concentrated small solute, may be pre-formed or may be self-forming under the influence of the centrifugal field. Gradient-forming solutes of choice for the self-forming gradient experiment are heavy salts such as chlorides, bromides, or iodides, as concentrated solutions of these have low viscosity, permitting (relatively) rapid gradient formation. As the density gradient is formed, each macrosolute migrates to form a band at the particular position in the density gradient corresponding to its buoyant density, i.e., that position where the applied centrifugal force is exactly cancelled by the buoyant force. The method works well, but suffers from two major disadvantages: [a] As conventionally performed the sample is centrifuged
until an equilibrium distribution of the gradient-forming
solute is achieved (up to 24 hours and sometimes longer) to assure the desired separation of macrosolutes. [b] Rotor velocity and hence speed of separation, are limited by the condition that the solution of the gradient-forming salt not attain saturation exceeded at the bottom (or outer edge) of the tubes at any time during centrifugation, so as to avoid the possibility of salt precipitation, which might overstress and damage the rotor or otherwise defeat the purpose of separation.
This disclosure is motivated by the desire to develop new kinds of time-saving density gradient separations and to optimize the conditions under which conventional density gradient separations are performed so as to obtain the desired separation in as short a time as possible.